1.Field of the Invention
This invention relates to an immunologically active fusion protein, useful in vaccination against dengue (DEN) virus as well as a diagnostic antigen. More particularly, this invention relates to a fusion protein antigen containing 366 amino acids of a maltose binding protein (MBP) from E. coli fused to 103 amino acids of the DEN envelope protein. This fusion protein antigen reacts with mouse monoclonal (3H5) and polyclonal (HIAF) antibody to dengue virus as well as human anti-DEN antibody, making it a useful antigen for diagnostic assays and when administered as a vaccine, can confer protective immunity to a substantial proportion of vaccinated subjects.
2.Description of the Prior Art
Prevention of DEN infections would alleviate a major health problem in tropical and subtropical areas of the world. Each of the four DEN serotypes may cause dengue fever and the more severe illnesses, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Infection with any one serotype of DEN appears to confer lifelong immunity to reinfection with the same serotype, but not to the other serotypes. Individuals sequentially infected with a different dengue serotype (secondary infection) appear to be at higher risk for developing more severe disease. Immmune enhancement has been proposed as the mechanism responsible for DHF/DSS. Because of this phenomenon, an effective vaccine must be either serotype specific or be against all four serotypes of dengue virus to insure that vaccination against a single serotype does not enhance infection with a different serotype.
Despite more than 50 years of effort, safe and effective vaccines have not been developed. Attempts to produce cell culture-derived live attenuated dengue vaccines by long term passage in primary kidney (PDK) cells were tried unsuccessfully (Ennis et al., J. Inf. Dis., 158:876-880, 1988). A dengue-1 candidate was shown to be genetically unstable (McKee et al., Am. J. Trop. Med. Hyg., 36(2):435-442, 1987). Other dengue 2, 3 and 4 candidates were also not suitable because they were either unstable, caused unmodified dengue fever or produced low levels of neutralizing antibody (Bancroft et al., J. Infect. Dis., 149:1005-1010, 1984, Hoke et al., Am. J. Trop. Med. Hyg., 43(2):219-226, 1990, Innis et al., Am. J. Trop. Med. Hyg., 40(6):676-687, 1989).
DEN viruses are composed of a single-stranded RNA molecule of positive polarity that is translated into three structural proteins, the capsid (C) protein, the membrane (M) protein, and the envelope (E) glycoprotein followed by seven nonstructural proteins (Henchal and Putnak, Clin. Microbiol. Rev., 3(4):376-396, 1990). The E protein is the major surface component of the virion membrane, it mediates virus binding to cell receptors and interacts with neutralizing antibody. Neutralizing antibodies are believed to correlate with protection (Henchal and Putnak, Clin. Microbiol. Rev., 3(4):376-396, 1990). Studies have shown that the E protein of DEN viruses contain cross reactive, subgroup specific, and serotype specific antigenic determinants and has been shown to elicit a protective immune response (Gentry et al., Am. J. Trop. Med. Hyg., 31:548-555, 1982, to Mc Cown et al., Am. J. Trop. Med. Hyg., 42(5):491-499, 1990). Immunization of rabbits with a purified native 33 kDa E protein fragment of the 17D Yellow Fever (17DYF) virus produced neutralizing antibodies and protection against intracerebral challenge (Brandriss et al., J. Enf. Dis., 161:1134-1139, 1990). Mice immunized with native purified DEN-2 envelope protein developed neutralizing antibodies and were protected against lethal virus challenge (Feighny et. al., Am. J. Trop. Med. Hyg., 47(4):405-412, 1992).
Several laboratories have reported production of recombinant DEN virus structural and nonstructural proteins in expression vectors including baculovirus, vaccinia virus and E. coli (Bray et. al., J. Virol., 63:2853-2856, 1989, Bray et al., J. Virol., 185:505-508, 1991, Lai et al, Vacc., 89:351-356, 1989, Mason et al., Virol., 158:361-372, 1987, Mason et al., J. Gen. Virol., 70:2037-2049, 1989, Men et al., J. Virol., 65:1400-1407, 1991, Putnak et al., Am. J. Trop. Med. Hyg.,45:159-167, 1991, Putnak et al., Virol., 163:93-103, 1988, Zhang et al., J. Virol. 62:3027-3031, 1988). Success with recombinant proteins as vaccines in the murine model has been variable. Some baculovirus recombinants have shown protection against virus challenge, but they were membrane-associated, making them difficult to purify without denaturation and were produced in low yields (Lai et al., Vacc., 89:351-356, 1989, Putnak et al., Virol., 163:93-103, 1988, Zhang et al., J. Virol., 62:3027-3031, 1988). As crude antigens they elicited low levels of neutralizing antibodies in mice, making them unlikely candidate vaccines. Vaccinia virus expressing the E protein and nonstructural protein one (NS1) used as a live recombinant virus failed to elicit a detectable seroresponse to either protein (Zhao et al., J. Virol., 62:4019-4022, 1987). An E. coli produced fusion protein with staphylococcal protein A containing 269 amino acids of the DEN-2 E protein and NS1 protein was recently reported to elicit neutralizing antibodies and protected mice against lethal challenge (Srivastava et al., Vacc., 13:1251-1258, 1995). Staphylococcal protein A however, has a high binding affinity for human immunoglobulin G, eliminating this fusion protein as a candidate vaccine (Kobatake et al., Anal. Biochem., 208(2):300-305, 1993). Recombinants containing the B domain of the E protein as a fusion protein with TrpE were expressed in E. coli as insoluble intracellular inclusion bodies and required detergents for its purification. These purified preparations did not elicit neutralizing antibodies in immunized mice nor did they confer protection against virus challenge (Fonseca et al., Am J. Trop. Med. Hyg., 44(5):500-508, 1991).